iNOS is essential to maintain a protective Th1/Th2 response and the production of cytokines/chemokines against Schistosoma japonicum infection in rats

Humans and a wide range of mammals are generally susceptible to Schistosoma infection, while some rodents such as Rattus rats and Microtus spp are not. We previously demonstrated that inherent high expression levels of nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS), plays an important role in blocking the growth and development of Schistosoma japonicum in wild-type rats. However, the potential regulatory effects of NO on the immune system and immune response to S. japonicum infection in rats are still unknown. In this study, we used iNOS-knockout (KO) rats to determine the role of iNOS-derived NO in the immune system and immunopathological responses to S. japonicum infection in rats. Our data showed that iNOS deficiency led to weakened immune activity against S. japonicum infection. This was characterized by the impaired T cell responses and a significant decrease in S. japonicum-elicited Th2/Th1 responses and cytokine and chemokine-producing capability in the infected iNOS-KO rats. Unlike iNOS-KO mice, Th1-associated cytokines were also decreased in the absence of iNOS in rats. In addition, a profile of pro-inflammatory and pro-fibrogenic cytokines was detected in serum associated with iNOS deficiency. The alterations in immune responses and cytokine patterns were correlated with a slower clearance of parasites, exacerbated granuloma formation, and fibrosis following S. japonicum infection in iNOS-KO rats. Furthermore, we have provided direct evidence that high levels of NO in rats can promote the development of pulmonary fibrosis induced by egg antigens of S. japonicum, but not inflammation, which was negatively correlated with the expression of TGF-β3. These studies are the first description of the immunological and pathological profiles in iNOS-KO rats infected with S. japonicum and demonstrate key differences between the responses found in mice. Our results significantly enhance our understanding of the immunoregulatory effects of NO on defensive and immunopathological responses in rats and the broader nature of resistance to pathogens such as S. japonicum.


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The data underlying the results presented in the study are available from (include the name of the third party and contact information or URL). This text is appropriate if the data are owned by a third party and authors do not have permission to share the data. However, the potential regulatory effects of NO on the immune system and immune response to S. japonicum infection in rats is still unknown. In this study, we used iNOS-knockout (KO) rats to determine the role of iNOS-derived NO in the immune system and immunopathological responses to S. japonicum infection in rats. Our data showed that iNOS deficiency led to weakened immune activity against S. japonicum infection. This was characterized by the impaired T cell responses and a significant decrease in S. japonicum-elicited Th2 responses and in cytokine and chemokine producing capability in the infected iNOS-KO rats. Unlike iNOS-KO mice, Th1associated cytokines were also decreased in the absence of iNOS in rats. In addition, a profile of pro-inflammatory and pro-fibrogenic cytokines was detected in serum associated with iNOS deficiency. The alterations in immune responses and cytokine patterns were correlated with a slower clearance of parasites, exacerbated granuloma formation and fibrosis following S. japonicum infection in iNOS-KO rats.
Furthermore, we have provided direct evidence that high levels of NO in rats can promote the development of pulmonary fibrosis induced by egg antigens of S.
japonicum, but not inflammation, which was negatively correlated with the expression of TGF-β3. These studies are the first description of the immunological and pathological profiles in iNOS-KO rats infected with S. japonicum and demonstrate key differences between the responses found in mice. Our results significantly enhance our understanding of the immunoregulatory effects of NO on defensive and immunopathological responses in rats and of the broader nature of resistance to pathogens such as S. japonicum. production. The Th2 response in rats seems to play an essential role in the protection of the host against Schistosoma. So far, the factors that lead to the different immune responses to Schistosoma infection in both hosts have not been demonstrated. In this study, our results show that an iNOS-dependent mechanism maintains the function of the immune system in rats by modulating CD4 + T cell-mediated Th1/Th2-associated cytokine responses and chemokine production. Additionally, the absence of iNOS led to slow clearance of parasites, increases in the development of worms and an exacerbation of granuloma formation and fibrosis in rats. Furthermore, high levels of NO in rats can promote the development of fibrosis induced by inflammation (rapid inflammatory repair). Therefore, this study demonstrates that the difference in iNOS levels between mice and rats is responsible for the different immune responses and outcomes induced by schistosome infection in both hosts.

Introduction
Schistosomiasis, is a zoonosis caused by the parasitic helminth Schistosoma. It is an important public health problem that affects around 290 million people in 78 countries [1]. The bulk of the morbidity and mortality of schistosomiasis ultimately results from the development of a Th2-driven inflammatory response and fibrosis in the host causing by Schistosoma eggs trapping in tissues, such as intestines and liver (intestinal schistosomiasis) and the urinary bladder wall (urogenital schistosomiasis) [2]. A wide range of mammals, including humans, are generally susceptible/permissive hosts of Schistosoma and develop chronic disease characterized by egg-granuloma formation and fibrosis after infection [3,4]. In contrast, the rat is a nonpermissive host in which the parasite does not cause typical egg granulomas in the liver of the infected host. This is because the rat is genetically resistant to Schistosoma infection and develops efficient immune responses to eliminate the worms [4] [5]. Typically, mice are widely used as a model of permissive host, for Schistosoma spp, to understand the immune responses during human schistosomiasis. During the initial stages of infection, mice display a Th1 immune response by producing high levels of Th1 cytokines (e.g., IFN-γ, interleukin (IL) -12, TNF-α), which may participate in protection against Schistosoma infection [6].
However, once Schistosoma eggs are produced at 5-6 weeks of infection, the host immune status dramatically shifts to a Th2 response, as shown by the increased production of Th2-associated cytokines IL-4, IL-5, IL-10, and IL-13 [7,8]. The Th2 response is involved in the development of egg-granuloma formation and fibrosis and is essential for host survival by protecting against inflammatory cytokines the mediate death during acute schistosomiasis. The excretory-secretory antigens (ES) from the eggs are powerful factors that induce Th2 polarization in schistosomiasis. A particularly striking difference, in the rat model of schistosomiasis compared to mice, is the fact that a Th2 response is driven in the absence of available egg production by worm pairs [4,9]. Therefore, it suggests that the, significantly greater, Th2 type response in rats seems to play an essential role in the protection of the host against Schistosoma [4]. Consequently, the different immune responses induced by schistosomes in mice and rats may be responsible for the different outcomes of host adaptability. However, the factors that lead to the different immune responses to Schistosoma infection in both hosts have not been elucidated. In previous work, we demonstrated a key role of nitric oxide in resistance to schistosome infection in rats suggesting its involvement in this process [10].
Nitric oxide (NO), produced from L-arginine by three isoforms of nitric oxide synthase (NOS), exhibits a variety of physiologic functions in mammals [11]. It is reported that NO plays an important role in regulating vascular function, neurotransmission, inflammatory responses, immune function, and host defense [12,13]. Inducible NOS (iNOS), expressed in response to proinflammatory cytokines (such as IFN-γ, TNF-a, and IL-1β) and/or microbial products (such as LPS), can rapidly produce large amounts of NO in contrast to the other two isoforms, the endothelial NOS (eNOS) and the neuronal NOS (nNOS) [12]. In previous studies, we have confirmed that the expression of iNOS and the levels of NO induced by stimulation of macrophages of rats are significantly higher than those in mice [10,14,15]. Furthermore, using iNOS knockout (iNOS-KO) rats, we have demonstrated that NO, inherently at high levels in rats, plays a crucial role in blocking S. japonicum growth, reproductive organ development, egg production, and the ability to lay fertilized eggs [10]. Furthermore, the inhibitory effect of NO acts by affecting mitochondrial respiration and energy production in this parasite [10]. Indeed, in addition to its direct cytotoxic effect on infectious pathogens in non-specific immune defense mechanisms, NO has also been found to be a potent immunoregulatory factor [16,17]. Previously, studies have shown that NO displays a significant immunosuppressive effect by causing inhibition of T cell proliferation, regulation of T-cell function [18][19][20] and inducing T helper cell deviation by suppression of Th1 (and Th2) cell responses [21,22]. Moreover, NO has also been demonstrated to influence the expression and function of many inflammatory factors [23]. Therefore, it raises the question as to whether the high expression of NO in rats affects the development of S. japonicum by regulating the immune response.
The majority of previous studies on the effects of NO on the immune system were based on mouse models. However, the effects of NO on the immune system vary with concentration, host species and response to different pathogens. For example, it was reported that high concentrations of NO could suppress the expansion of Th1 cells by inhibiting IL-12 synthesis, whereas low doses of NO selectively promoted Th1 cell differentiation and had no effect on Th2 cells [21]. Furthermore, NO has been reported to preferentially down-regulate Th1-mediated immune responses in the murine system [24,25], however, this selective inhibitory effect on Th1 responses was not found in activated human T cells [22]. Additionally, NO induces different immune responses to different pathogens in the same host. It is shown that a Th1 immune response was enhanced in iNOS-KO mice after infection with Leishmania major [26], whereas a gastric Helicobacter infection displayed a reduction of the Th1 response in iNOS-KO mice, relative to that of the WT mice [27]. Thus far, little is known in rats about the regulatory role of NO on the immune system and the outcomes of iNOS gene deficiency on the evolving immune response. It is possible that the differences in NO levels between mice and rats [14] may be linked to differences in Schistosoma infection-induced immune responses. Therefore, there is a need to investigate the role of NO in the regulation of the immune response that inhibits Schistosoma infection in rats.
In the present study, using S. japonicum infected iNOS-KO and wild-type rat models, we investigated the effects of iNOS-derived NO on immunity and immunopathological responses to S. japonicum infection in rats. Our results show that an iNOS-dependent mechanism maintains the function of the immune system in rats by modulating CD4 + T cell-mediated Th1/Th2-associated cytokine responses and chemokine production. This plays a crucial role in host protective immunity against S. japonicum infection in rats. Thus, this study significantly enhances our understanding of the immunoregulatory effects of NO on defensive and immunopathological responses in rats. Hepatic granulomatous inflammation and fibrosis is the primary cause of chronic morbidity in schistosomiasis. Strikingly, the infected KO rats developed significant granulomatous inflammation and fibrosis in the liver, particularly at the chronic time point of 12 weeks post-infection, compared with infected WT rats (Fig. 1C). Dead worms in the liver sections were clearly observed in the infected KO rats (Fig. 1C).

Results
Moreover, infected KO rats experienced significantly greater weight loss than infected WT rats, especially after 4 weeks post-infection (P＜0.01) (Fig. 1D). When infected with a low dose of parasites, no death was found both in WT or KO rats.
However, at high doses, the survival of iNOS-KO rats was dramatically decreased and ultimately 100% of animals succumbed between days 11-34 post-infection, while no mortality was observed in the WT control group during this period (Fig. 1E).
Together, these results showed that iNOS-mediated functions are essential in host protection against S. japonicum infection in rats, as characterized by accelerating clearance of parasites, alleviating hepatic pathological responses and promoting host survival.

iNOS deficiency led to a decrease in the frequency of T cells and an increase in B cells in rats infected with S. japonicum
In order to determine whether iNOS is involved in anti-Schistosoma infective immunity by affecting the profile of immune cells in rats, we examined the differences in frequencies of T cells and B cells in spleens, mesenteric lymph nodes (LN), and liver cells by comparing infected iNOS-KO rats and WT rats. In comparison with WT rats, the results showed that there was a lower frequency of and liver (0.18, P＜0.05) following infected with S. japonicum (Fig.2E). Surprisingly, however, the decreased CD4 + /CD8 + ratio was also not found in the blood, spleen and LN of KO rats following infection with S. japonicum (Fig.2E). Thus, the results reveal that NO is necessary for maintaining the host's cellular immune defense against infection in wild-type rats.

A profile of pro-inflammatory and pro-fibrogenic cytokines developed in iNOS deficient rats with schistosomiasis
To investigate the systemic immune response developed in KO rats, cytokine-  [26,27]. In addition, the immunosuppressive cytokine IL-10 was also significantly downregulated in KO rats, which displayed a notable significant difference at 7 weeks postinfection between both groups (Fig. 3D). Expression of IL-17 was not detectable (data not shown). Thus, it suggests that a pro-inflammatory and pro-fibrogenic immunological environment developed with iNOS deficiency in infected rats.

iNOS expression ensures maximal development of Th2 and Th1 responses in the rat during S. japonicum infection
Previous studies have reported that a Th2 response was strongly activated in the absence of available egg production in the rat model of schistosomiasis and speculated that the increased Th2 response was involved in the resistance to Schistosoma infection [4,9]. We also observed increased levels of the Th2-related cytokines IL-4 and IL-5 in serum from infected WT rats. Therefore, to determine whether the high levels of expression of iNOS were responsible for the elevated Th2 response in rats following infection with S. japonicum, Th1 and Th2 responses were compared between WT and iNOS-KO rats. Here, Th1 and Th2 responses were analyzed by the expression of IFN-γ and IL-4 in CD4 + T cells from peripheral blood, LN, and spleen from 7-week S. japonicum-infected animals detected by flow cytometry. As shown in Fig.4A and 4B, the frequency of CD4 + T cells producing IL-4 was slightly increased in the blood (P＜0.05) of naïve KO rats, compared with naïve WT rats. As expected, there was a marked increase in the frequency of IL-4producing CD4 + T cells in the blood (P＜0.001), LN (P＜0.01), and spleens (P＜ 0.001) of WT rats following infection with S. japonicum. However, although the frequency of IL-4-producing CD4 + T cells slightly increased in infected KO rats, when compared to naïve KO rats, no significant differences were found in LN and spleens (P＞0.05) although these were lower than those found in the infected WT rats. The results suggest an impaired Th2 response in infected iNOS-KO rats. The reduction in the Th2 response did not result from an increased Th1 response because the frequency of CD4 + T cells producing IFN-γ was also decreased in the blood, LN, and spleens of infected KO rats, compared with infected WT rats (P＞0.05) ( Fig.4C and 4D). The results suggest that high expression levels of iNOS are required to sustain CD4 + T cell responses in rats with schistosomiasis.

Cytokine and chemokine production in local tissues (liver) is dependent on the expression of iNOS in rats during S. japonicum infection
The liver is the main location of pathological changes and schistosome parasitism in rats with schistosomiasis. To evaluate the role of iNOS in regulating the chemotaxis and effector effects of immune cells involved in immunopathology and activity against Schistosoma, we compared the immunological characteristics of the livers of WT and KO rats. As shown in Fig.5A, local (liver) IFN-γ, IL-4, IL-10, TGF-β1 levels in infected KO rats were significantly lower than those in infected WT rats (P＜0.05), which was consistent with the results found for serum (Fig. 3).
Surprisingly, the expression of IL-6 mRNA also showed marked reduction in infected KO rats (P＜0.01), in contrast to the serum which showed significantly increased expression (Fig. 3A). Again, similar findings were confirmed by immunohistochemistry, which showed that the expression of IFN-γ, IL-10, TGF-β3, and IL-6 at the periphery of granulomas in KO rats were significantly lower than those in WT rats (Fig.5B). Furthermore, the expression of the monocyte chemoattractant protein (MCP)-1, Eotaxin, Eotaxin-2, neutrophil-activating protein (NAP)-3 (also named CXCL1), macrophage inflammatory protein (MIP)-2a (also named CXCL2) were significantly decreased in the liver of infected KO rats, indicating a decline in the ability to recruit immune cells to the inflammatory site iNOS also participates in promoting pulmonary granuloma associated fibrotic processes, but not inflammation, in wild type rats To determine the possible immunoregulatory effects of iNOS in formation and development of fibrosis in rats, we exploited the schistosome egg-induced pulmonary granuloma model [28]. Pulmonary granuloma associated fibrosis was compared in the WT and iNOS-KO rats on day 7 and 14 post-challenge, following intravenous injection with 15,000 live eggs. We have described previously that there were no significant differences in granuloma size in both genotypes of animals on day 7 and 14 post-challenge, although the KO rats displayed a, non-significant, slight decrease [10]. When pulmonary granuloma-associated fibrosis in WT and KO rats were compared by Masson's trichrome staining (Fig. 6A and 6B), surprisingly, compared with WT rats, the KO rats showed an average of 78% increase in blue fibrosis staining on day 7 (P＜0.01), the peak of the granulomatous response [29], while displaying a slight, non-significant, reduction (27%) in collagen deposition on day 14 postchallenge (P＞0.05). The results showed that iNOS in rats inhibited the formation of fibrosis in the acute phase and promoted fibrosis in the chronic phase. Furthermore, in comparing the fibrotic process in KO and WT rats, WT rats displayed a slight increase in collagen deposition at day 14 compared with day 7 post-challenge. In contrast, collagen deposition was markedly decreased in KO animals at day 14, with nearly 56% less than that found at day 7 post-challenge (P＜0.01) (Fig. 6B). In addition, fibrosis was also examined using immunohistochemistry with alpha-smooth muscle actin antibodies (α-SMA), a marker of activated myofibroblasts, as shown in Figures 6C and 6D. Consistent with the collagen deposition by Masson's trichrome, KO rats also displayed much stronger staining for α-SMA at day 7 post-challenge and weakened at day 14 post-challenge ( Figure 6C and 6D). The results showed that the fibrosis process is inhibited in KO rats at day 14 post-challenge, compared with WT rats which showed persistently elevated α-SMA expression. Therefore, these results suggest that iNOS accelerates the pulmonary granuloma-associated fibrotic process in rats. On the other hand, importantly, our results illustrated that the exacerbated granuloma-associated inflammation and fibrosis in the liver of iNOS-KO rats were attributed to the increased worm burden, egg load, and viability, but not the effects of iNOS on pathology.
To dissect the possible mechanisms for iNOS-mediated promoting pulmonary granuloma-associated fibrotic process in rats, we detected the expression of cytokines at the periphery of granulomas in WT and KO models by immunohistochemistry. As shown in Fig.6E and 6F, the KO rats displayed a slight, non-significant, reduction in the expression of IFN-γ and IL-10 and a slight, non-significant, increase in the expression of IL-6 and TNF-α at day 7 and 14 in comparison with WT rats (P＞0.05). This is consistent with the results found in the serum of KO rats infected with S. japonicum (Fig.3). Interestingly, compared with WT rats, however, the expression of TGF-β3 around pulmonary granulomas in KO rats showed nearly a 64% reduction on day 7 (P＜0.01) and 67% increase on day 14 post-challenge (P＜0.05) (Fig.6E and   6F), which is opposite to the trend observed in pulmonary granuloma associated fibrosis. To further investigate whether the expression of TGF-β3 at the periphery of granulomas in lungs was associated with the development of fibrosis in rats, we conducted a correlation analysis between the expression level of TGF-β3 around pulmonary granuloma and pulmonary granuloma associated fibrosis in WT and KO rats. Figure 6G shows that the TGF-β3 levels were negatively correlated with fibrosis quantified using Masson's Trichrome in WT and KO rats (r = −0.55; P= 0.035). No significant correlation was found between TGF-β3 levels and the expression of α-SMA at the periphery of granulomas (r = −0.46; P= 0.082) ( Figure 6H). The results indicate that TGF-β3 may be involved in the iNOS-mediated pathway promoting the pulmonary fibrotic process in rats.

Discussion
The rat, a non-permissive host, develops immune responses directed at juvenile or adult Schistosoma that causes rapid elimination of all parasites and no typical egggranuloma formation [4,5], while mice (permissive hosts) do not. Interestingly, schistosome eggs elicit a dominant Th2 immune response within mouse hosts [8,30], whereas rats with schistosomiasis develop significant a Th2 response in the absence of available egg production [4,9]. Our previous studies have demonstrated that the expression of iNOS and the levels of NO in peritoneal macrophages of rats are significantly higher than those in mice [10,14,15]. In the present study, our results showed that, following infection with S. japonicum, iNOS gene deficiency in rats led to slow clearance of parasites, increases in the development of worms and an exacerbation of granuloma formation and fibrosis. Importantly, S. japonicum-elicited a Th2 response that was significantly impaired in iNOS-KO rats. Therefore, this study demonstrates that the difference in iNOS levels between mice and rats is responsible for the different immune responses and outcomes induced by schistosome infection in both hosts.
The results presented here show that the worm burden collected from the hepatic portal vein infusion of infected iNOS-KO rats was significantly increased compared with the infected WT rats at week 1 and 7 post-infection, while no significant differences were found at week 4 post-infection. Coulson, et al. [31] have concluded from their results that the effector response of NO, against an embolized parasite in the lungs, acts by blocking further migration rather than by direct cytotoxic killing.
Therefore, in our study, the elevated worm load, at week 4, in WT rats may be attributed to the parasites that remained in the lungs having reached the liver. This is due to the migration of S. japonicum from the lung to the liver being inhibited by NO in WT rats. This inference was further supported by the observations that the spines on the tegument in the middle part of the body of the schistosomula from iNOS-KO rats were much reduced compared to those found in WT rats (Fig. S2), that the loss of spines contributes to reducing the resistance during their migration [32]. Furthermore, NO in rats promotes the clearance of S. japonicum mainly after 4 weeks postinfection, such that the worm load decreased most obviously at 7 weeks after infection, which implies that it is in the liver rather than in the lungs that NO is effective in eliminating the parasite. This opinion is supported by the results reported by Wilson et al. [31] that NO was not the major agent causing the pulmonary effector response to eliminate S. mansoni in mice exposed to the radiation-attenuated vaccine.
It is worth noting that the iNOS-KO rats died at a significantly accelerated rate, with all animals succumbing within 5 weeks infected with a high dose of parasites, compared with WT rats. The significantly increased mortality may attribute to the increased parasite burden and development-mediated inflammation, as demonstrated by the significant increase in pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) levels in the serum of infected iNOS-KO rats within 5 weeks post-infection.
Based on the results, we deduce that S. japonicum-infected iNOS-KO rats are immunocompromised and unable to clear the infection. Thus, we first examined whether the generation of immune cells was affected by iNOS deficiency. Our results showed that iNOS deficiency in rats led to a reduction in the frequencies of T cells and an increase in B cell numbers in mesenteric lymph node and livers, but having no effects on macrophages. These results demonstrate that the expression of iNOS plays an important role in maintaining the host T-cell immune response and inhibiting B cell populations in rats with schistosomiasis. T cells play an essential role in the immune response to schistosomiasis. It has been reported that the preferential proliferation of T cells in the draining lymph nodes of mice exposed to S. mansoni is central to the induction of protective immunity [33,34]. In contrast, the role of B cells in the process of schistosome infection seems to be less important. Ferru et al. [35] demonstrated that B lymphocytes were not essential for the development of the general immune response towards S. mansoni in the mouse, they showed that the absence of B cells in mice did not affect the adult worm and egg burdens in comparison to control groups. Thus, it indicates that iNOS-mediated T-cell immune responses may play an important role in host protective immunity against S. japonicum in rats. CD4 + and CD8 + T cells are important for regulating the host's immune function against infection [36]. CD4 + T cells participate in modulating the activity of the immune response mainly by regulating the secretion of cytokines and antibodies [37], and CD8 + T cells take part in the cytotoxic effect [38]. The CD4 + /CD8 + ratio is one of the indicators for assessing host cellular immune status, a decline in the CD4 + /CD8 + ratio implies weakening immune function of the host [36].
In our study, a decrease in CD4 + T cells and CD4 + /CD8 + ratio was observed in naïve iNOS-KO rats compared to WT rats, which indicated a likely decline in immune response in iNOS-KO rats. Furthermore, the increase in CD4 + T cells and CD4 + /CD8 + ratio in iNOS-KO rats following infection with S. japonicum means that this parasite infection can activate and enhance the host cellular immune functions in iNOS-KO rats. A previous study has shown that immunosuppression in murine schistosomiasis may be caused by the activation of CD8 + T-cell function, which suppresses the maturation of CD4 + T cell subsets [39]. Thus, the results reveal that NO is necessary for maintaining the host's cellular immune defense against S. japonicum in wild-type rats.
Previous studies have reported that the Th2 response plays an important role in resistance to Schistosoma infection in rats, that the worm burden was significantly increased in rats following treatment with anti-IL-4R or anti-IL-13R antibodies and which could be associated with their ability to control the production of IgE and eosinophils [4]. In addition, previous results revealed that the immune effector mechanism against schistosomula in the lungs requires CD4 + T cells capable of producing IFN-γ in mice [31,34]. Therefore, we compared the S. japonicum-elicited Th1 and Th2 responses in iNOS-KO rats and WT rats. Interestingly, in infected iNOS-KO rats, the frequency of IL-4-producing CD4 + T cells was impaired in the mesenteric lymph nodes and spleen, and the levels of Th2-associated cytokines (IL-4 and IL-5) in the serum and liver were also markedly reduced, compared with infected WT rats. Surprisingly, the reduction in Th2 response did not result from an increased Th1 response, because the frequency of CD4 + T cells producing IFN-γ was also obviously decreased in the lymph nodes, spleens, and liver of infected iNOS-KO rats comparison to the WT rats. Therefore, the results indicate that iNOS expression ensures maximal development of Th2 and Th1 responses in the rat during Schistosoma infection. The downregulated Th2 response and IFN-γ levels in iNOS-KO rats are not conducive to the elimination of parasites. Interestingly, these results found in infected iNOS-KO rats differ from the observations obtained from infected iNOS-KO mice, which develop significantly enhanced Th1-associated cytokine responses (IFN-γ) and lower expression of Th2-associated cytokines (IL-4, IL-5) levels in the lungs after vaccination with attenuated S. mansoni [40] and, furthermore, displayed partially reduced resistance [31,40]. Similar Th-associated cytokine response profiles were also found in the egg/IL-12-sensitized iNOS-KO mice, while there was little or no significant effect on the Th2 or Th1-type cytokine polarization during the natural course of infection with S. mansoni in the iNOS-KO mice versus WT mice, given the low expression of iNOS detected in infected mice [41]. These data indicate that iNOS activation is not essential for Th1 response development in mice. Thus, the results obtained with iNOS-deficient animals (rats and mice) accurately reflect that the difference in NO concentration in the host could lead to different levels of resistance to S. japonicum by regulating the immune response of the host.
Moreover, it is of note that the transcriptional levels of all cytokines and chemokines measured in the liver (the main location of schistosome parasitism in rats) of iNOS-KO rats were markedly lower than those of WT rats. Chemokines play an important role in regulating cell trafficking. When the parasites persist in a particular organ, the chemotactic signals facilitate the recruitment of macrophages, eosinophils, basophils, and T cells to form a granuloma to keep the parasites or eggs sequestered under control [42]. Therefore, the down-regulated chemokines in iNOS-KO rats are not conducive to the recruitment of inflammatory cells to the periphery of worms and therefore were unable to secrete abundant cytokines necessary for rapid elimination of Schistosoma. These results demonstrate that both the cytokine and chemokine producing capabilities are ensured by the expression of iNOS in rats. This notion is consistent with the previous findings in iNOS-KO mice that the size of inflammatory foci around schistosomula in the lungs was diminished [40]. In addition, a proinflammatory immune environment was developed in naïve iNOS-deficient rats, with an increase in TNF-α, IL-1β, and IL-6 expression, compared with naïve WT rats.
Previous studies have demonstrated that pro-inflammatory stimuli could promote schistosome development and exacerbate infection (44). A similar observation was also demonstrated by Riner et al (45), they revealed that repeated administration of an endogenous inflammatory stimulus could restore schistosome development in recombination activating gene-deficient (RAG -/-) mice, which fail to induce liver inflammation and necrosis after infection with S. mansoni. In addition, some studies showed that TNF could indirectly stimulate schistosome development by binding to the TNF receptor and TLR signaling pathway (46)(47)(48)(49). Thus, based on our findings, we demonstrate that the high levels of expression of NO in rats can inhibit the development of S. japonicum by regulating the host immune system.
The role of iNOS in fibrosis is highly controversial, with some studies showing pathogenic [43][44][45] and protective [41,46] roles for this enzyme. Notably, in our present study, the increased fibrosis in the liver of infected iNOS-KO rats was attributed to the marked increased egg production and the viability of parasite eggs [10], which formed larger sized granulomas and developed into more severe fibrosis in comparison to infected WT rats. Therefore, we could not determine the protective effect of NO on fibrosis in rats. With the schistosome egg-induced pulmonary granuloma model, interestingly, we found that iNOS played different roles during acute versus chronic stages. Pulmonary granuloma-associated fibrosis was inhibited at the acute stage (7 days post-challenge) by the expression of iNOS in rats, while such protection was completely abolished during the chronic stage (14 days postchallenge). In other words, these results suggest that the expression of iNOS accelerates the pulmonary granuloma-associated fibrotic process in rats. These observations were consistent with the results reported in allergen exposure-induced pulmonary fibrosis models that fibrosis was completely absent in iNOS-KO mice when using the chronic protocol [47]. The reason for this result may be that the iNOS gene deletion caused a failure to induce the infiltration of fibroblasts and collagen production in the chronic stage. While the presence of iNOS promoted the development of fibrosis, induced by inflammation, and suggests a possible cause of rapid inflammatory repair in the lungs and liver of rats with schistosomiasis. Studies in a wide range of experimental models have demonstrated the profibrotic actions of TGF-β1 [48][49][50][51]. In contrast, some experimental studies indicated that TGF-β2 and TGF-β3 can exert antifibrotic effects [52][53][54]. In the present study, the result showed that the effect of iNOS on egg-induced lung fibrosis was negatively correlated with the expression of TGF-β3, which suggests that TGF-β3 may protect against the development of pulmonary fibrosis. This idea confirms earlier studies showing that TGF-β3 decelerates the progress of radiation-induced pulmonary fibrosis by hindering fibrocyte recruitment and regulating the IFN-γ/IL-4 balance [53].
In conclusion, these results reveal that iNOS plays a critical role in the regulation of immune system reactivity, particularly in Th1/Th2-associated cytokine responses and chemokine production in rats, during S. japonicum infection, which leads to a rapid rejection of schistosome survival and no granuloma formation. Furthermore, we provide direct evidence that high levels of NO in rats can promote the development of fibrosis, induced by inflammation, and suggests a possible cause of rapid inflammatory repair in the lungs and liver of rats with schistosomiasis. The key interactions, reported in this study, are schematically represented in Figure 7. Thus, this study significantly enhances our understanding of the immunoregulatory effect of NO on defense and immunopathological responses in rats.

Ethics statement
All animal work was approved by the Laboratory Animal Use and Care

Animals and parasite infections
Sprague Dawley (SD) rats were purchased from the Medical Experimental Animal Center of Guangdong Province. iNOS-KO rats were generated with the transcription activator-like effector nuclease technology (TALENs) [55]. These deficient rats are viable, fertile and do not display any obvious appearance or physical abnormalities. Animals were kept in ventilated cages with available food and water under specific-pathogen-free conditions in the Sun Yat-Sen University following the university policy. Six to eight week old male rats were used for the study.

Histopathology and fibrosis
Pulmonary and liver tissues were fixed in 4% neutral buffered formalin, embedded in paraffin for sectioning, dewaxed, and stained with Masson's trichrome for fibrosis analysis. Collagen is shown as blue in Masson staining, and the blue area reflects the amount of collagen. The severity of fibrosis was determined by the area of collagen using Image-Pro Plus software. All sections were imaged using an automatic slide scanning system (AxioScan.Z1, Zeiss, Germany) at 10X magnification.

Immunohistochemistry
For immunohistochemical analysis, the dewaxed sections were boiled in 10 mmol/L citrate buffer for 20 min for epitope retrieval following washing three times in PBS. After incubating in 3% hydrogen peroxide, the sections were blocked with 1% BSA for 1 hour. The slices were incubated with anti-rat antibodies against IL-10

Cytokine analysis
The cytokines in serum samples were assayed using a rat 14plex cytokine kit (eBioscience, CA) according to the manufacturer's instructions. Samples were read using a Bio-Plex Suspension Array System (Biorad, USA).

RNA isolation and Real-time PCR
Liver tissues were placed in a 500ul Trizol reagent (Invitrogen, USA) and mashed with TissueLyser II (Qiagen, USA). Total RNA was isolated and further purified using RNeasy Mini Kit (Qiagen, USA) following the manufacturer's instructions. The concentration of RNA was quantified using a NanoDrop ND-1000 spectrophotometer (Nanodrop, USA). First-strand cDNA was synthesized using isolated RNA, Superscript II reverse transcriptase (Invitrogen, USA), and oligo dT as a primer.

Scanning Electron Microscopy.
Parasites collected from infected mice, WT, and iNOS-KO rats at 7-day postinfection were fixed individually with 0.2M PBS containing 2.5% glutaraldehyde (pH 7.4) at 4 °C for 24 h. After washing three times with PBS and six times with distilled water, the samples were dehydrated in gradient ethanol and ethanol exchanged with acetone and isoamyl acetate. The tegument of the schistosomula was observed and photographed using a scanning electron microscope (S-2500, Hitachi) following critical point-drying and being coated with gold.

Statistical analysis
All statistical analyses were performed using SPSS 19 software. Significant differences between the two groups were determined using a student's unpaired T-test with Welch's correction or one-way ANOVA. Pearson correlation was used for correlation analysis. Graphs and analyses were performed using GraphPad Prism version 7.00 for Windows, San Diego, CA, USA. All data shown are presented as the mean ± SEM, and P values ≤0.05 were considered statistically significant.
Data Availability Statement: All relevant data are within the manuscript and its supporting information files.        Expression is normalized to β-actin. Data shown are mean ± SEM and repeated twice with similar results, n = 5 rats per group. Significant differences have been noted, * P < 0.05, ** P < 0.01, *** P ＜0.001. (B) Hepatic granuloma-associated cytokine expression was detected by immunohistochemistry (IHC). Brown (arrow) indicates a positive signal (Scale bars: 20 μm).  associated consequences: CD3 + T cells and CD4 + T cells were depleted, the ratio of CD4 + / CD8 + decreased, Th2 and Th1 immune cells upregulated, and the secretion of cytokines and chemokines increased. These immune effects mediated by iNOS lead to rapid elimination of schistosome, inhibition of worm development and no typical egggranuloma formation in rats. Therefore, schistosomiasis does not cause death in rats.
However, T cell responses were impaired in the absence of iNOS in rats, which resulted in delayed clearance of worms, increasing the development and oviposition of schistosomes, a lot of egg-granuloma formation which developed into fibrosis, and produced a lot of inflammatory cytokines, eventually leading to the death of the host.